TDP-43 pathology was first identified in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but was later found in other incurable neurological disorders including Alzheimer's disease (AD). It is characterized by decreased solubility, hyper-phosphorylation and abnormal accumulation of TDP-43 in the cytosolic compartment, which results in its nuclear depletion. Despite considerable effort to investigate the function of TDP-43, we still have a very poor understanding of the molecular events underlying TDP-43 pathogenesis. For instance, little is known about the molecular targets and mechanisms mediating its pathological cascade, and which and how many pathways orchestrate the disease. Additionally, recent studies indicate that TDP-43 may also play a role in AD pathogenesis by regulating A?42 plaque formation and tau aggregation. A key challenge, therefore, is to identify critical proteins and pathways capable of blocking TDP- 43 toxicity and its potential interactions with concurrent A?42 and tau pathologies. To address this, we recently performed an unbiased loss-of-function screen searching for modifiers of mutant TDP-43 toxicity in the Drosophila eye. To do so, we used a next generation collection of 6,261 RNA lines against human homologues and found 375 modifiers, including more than 100 suppressors. Our hypothesis is that a comprehensive functional analysis of these modifiers, including therapeutic challenges in other neurodegenerative models, will help to deconstruct and identify the most relevant pathological cascades. Therefore, this application aims at validating, classifying and prioritizing the best TDP-43 suppressors among this constellation of modifiers (Aim 1). Additionally, we will define the therapeutic potential of the top candidates and their combinations in additional models of FTLD, ALS and AD (Aim 2). Since co-manipulation of TDP-43, A?42 and tau in vivo is very difficult to study due to the aggressive synergistic toxicity between these factors, we developed a new optogenetic expression system to bypass this limitation. This new system allows unprecedented spatiotemporal control of gene expression in response to light quantity, duration and direction and, thus, we will use it to substantially increase knowledge of how these proteins interact using in vivo and ex vivo paradigms (Aim 3). Taken together, these experiments are highly significant because they will tease apart the complex mechanisms mediating TDP-43 proteinopathies and will provide valuable information for the development of new therapeutic strategies. In addition, we will provide new optogenetic tools for the simultaneous analysis of multiple neurotoxic proteins with unprecedented resolution.